Obesity represents a key risk and prognostic factor for breast cancer, which most commonly presents as hormone receptor positive (HR+) disease in postmenopausal women. However, the molecular mechanisms underlying obesity-driven breast cancer remain poorly understood, which is particularly alarming given the obesity epidemic in the U.S. Hypoxia, altered extracellular matrix (ECM) deposition, and elevated stromal estrogen are characteristic of the obese microenvironment, but whether or not the integrated effects of these parameters functionally corroborate towards obesity-dependent breast tumorigenesis remains unclear. In obese adipose tissue, hypoxia is caused by diffusion-limited oxygen transport, which induces oxidative stress that can enhance fibrotic tissue remodeling via increasing myofibroblast differentiation. Myofibroblasts, in turn, elevate ECM stiffness, which not only perturbs epithelial morphogenesis, but can also impact macrophage recruitment and activation. Indeed, macrophage activation is elevated in obese vs. lean adipose tissue and enhance breast cancer risk by stimulating aromatase expression, an enzyme that catalyzes the conversion of androgen precursors to estrogens. Nevertheless, it remains unknown whether obesity-induced hypoxia promotes tumorigenesis via stiffness-dependent macrophage activation, and ultimately, enhanced estrogen synthesis. This project will investigate the overall hypothesis that hypoxia increases interstitial ECM stiffness in obese adipose tissue by elevating myofibroblast differentiation and that the resulting changes in ECM physicochemical properties promote malignancy (i) via direct effects on tumor cells and (ii) by altering macrophage-dependent induction of stromal aromatase expression and activity. To address this hypothesis, three specific aims will be pursued. Aim 1: Determine the ability of obesity-associated hypoxia to enhance interstitial ECM stiffness of mammary adipose tissue. Aim 2: Characterize the effects of increased ECM stiffness on HR+ mammary tumor cells and determine the ability of macrophages to enhance stromal aromatase. Aim 3: Assess the integrated effects of increased ECM stiffness and elevated stromal estrogen on HR+ tumorigenesis, and evaluate whether therapeutic reduction of stiffness can inhibit this process. These studies will be conducted using a multidisciplinary approach that leverages the engineering, biology, and clinical expertise of the investigative team. Collectively, the findings should establish a functional link between obesity and stiffness-dependent tumorigenesis of HR+ breast cancer, and motivate novel therapeutic strategies that interfere with these pathways. Insights gained by this project should also offer broad relevance to other non-breast cancers whose prevalence is similarly enhanced with obesity.